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Title: Determination and Comparison of the Baseline Proteomes of the Versatile Microbe Rhodopseudomonas palustris under its Major Metabolic States

Abstract

Rhodopseudomonas palustris is a purple nonsulfur anoxygenic phototrophic bacterium that is ubiquitous in soil and water. R. palustris is metabolically versatile with respect to energy generation and carbon and nitrogen metabolism. We have characterized and compared the baseline proteome of a R. palustris wild-type strain grown under six metabolic conditions. The methodology for proteome analysis involved protein fractionation by centrifugation, subsequent digestion with trypsin, and analysis of peptides by liquid chromatography coupled with tandem mass spectrometry. Using these methods, we identified 1664 proteins out of 4836 predicted proteins with conservative filtering constraints. A total of 107 novel hypothetical proteins and 218 conserved hypothetical proteins were detected. Qualitative analyses revealed over 311 proteins exhibiting marked differences between conditions, many of these being hypothetical or conserved hypothetical proteins showing strong correlations with different metabolic modes. For example, five proteins encoded by genes from a novel operon appeared only after anaerobic growth with no evidence of these proteins in extracts of aerobically grown cells. Proteins known to be associated with specialized growth states such as nitrogen fixation, photoautotrophic, or growth on benzoate, were observed to be up-regulated under those states.

Authors:
 [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [2];  [1];  [1];  [3];  [4];  [5];  [6];  [1]
  1. ORNL
  2. {Greg} B [ORNL
  3. University of British Columbia, Vancouver
  4. University of Washington, Seattle
  5. Ohio State University
  6. {Bob} L [ORNL
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
978060
DOE Contract Number:
DE-AC05-00OR22725
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Proteome Research; Journal Volume: 5; Journal Issue: 2
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 54 ENVIRONMENTAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; CARBON; CENTRIFUGATION; CHROMATOGRAPHY; DIGESTION; FRACTIONATION; GENES; MASS SPECTROSCOPY; METABOLISM; NITROGEN; NITROGEN FIXATION; PEPTIDES; PROTEINS; RHODOPSEUDOMONAS; SOILS; STRAINS; TRYPSIN; WATER

Citation Formats

Verberkmoes, Nathan C, Shah, Manesh B, Lankford, Patricia K, Pelletier, Dale A, Strader, Michael B, Tabb, Dave L, McDonald, W Hayes, Barton, John W, Hurst, Gregory, Hauser, Loren John, Davison, Brian H, Beatty, Thomas, Harwood, Caroline S, Tabita, F Robert, Hettich, Robert, and Larimer, Frank W. Determination and Comparison of the Baseline Proteomes of the Versatile Microbe Rhodopseudomonas palustris under its Major Metabolic States. United States: N. p., 2006. Web. doi:10.1021/pr0503230.
Verberkmoes, Nathan C, Shah, Manesh B, Lankford, Patricia K, Pelletier, Dale A, Strader, Michael B, Tabb, Dave L, McDonald, W Hayes, Barton, John W, Hurst, Gregory, Hauser, Loren John, Davison, Brian H, Beatty, Thomas, Harwood, Caroline S, Tabita, F Robert, Hettich, Robert, & Larimer, Frank W. Determination and Comparison of the Baseline Proteomes of the Versatile Microbe Rhodopseudomonas palustris under its Major Metabolic States. United States. doi:10.1021/pr0503230.
Verberkmoes, Nathan C, Shah, Manesh B, Lankford, Patricia K, Pelletier, Dale A, Strader, Michael B, Tabb, Dave L, McDonald, W Hayes, Barton, John W, Hurst, Gregory, Hauser, Loren John, Davison, Brian H, Beatty, Thomas, Harwood, Caroline S, Tabita, F Robert, Hettich, Robert, and Larimer, Frank W. Sun . "Determination and Comparison of the Baseline Proteomes of the Versatile Microbe Rhodopseudomonas palustris under its Major Metabolic States". United States. doi:10.1021/pr0503230.
@article{osti_978060,
title = {Determination and Comparison of the Baseline Proteomes of the Versatile Microbe Rhodopseudomonas palustris under its Major Metabolic States},
author = {Verberkmoes, Nathan C and Shah, Manesh B and Lankford, Patricia K and Pelletier, Dale A and Strader, Michael B and Tabb, Dave L and McDonald, W Hayes and Barton, John W and Hurst, Gregory and Hauser, Loren John and Davison, Brian H and Beatty, Thomas and Harwood, Caroline S and Tabita, F Robert and Hettich, Robert and Larimer, Frank W},
abstractNote = {Rhodopseudomonas palustris is a purple nonsulfur anoxygenic phototrophic bacterium that is ubiquitous in soil and water. R. palustris is metabolically versatile with respect to energy generation and carbon and nitrogen metabolism. We have characterized and compared the baseline proteome of a R. palustris wild-type strain grown under six metabolic conditions. The methodology for proteome analysis involved protein fractionation by centrifugation, subsequent digestion with trypsin, and analysis of peptides by liquid chromatography coupled with tandem mass spectrometry. Using these methods, we identified 1664 proteins out of 4836 predicted proteins with conservative filtering constraints. A total of 107 novel hypothetical proteins and 218 conserved hypothetical proteins were detected. Qualitative analyses revealed over 311 proteins exhibiting marked differences between conditions, many of these being hypothetical or conserved hypothetical proteins showing strong correlations with different metabolic modes. For example, five proteins encoded by genes from a novel operon appeared only after anaerobic growth with no evidence of these proteins in extracts of aerobically grown cells. Proteins known to be associated with specialized growth states such as nitrogen fixation, photoautotrophic, or growth on benzoate, were observed to be up-regulated under those states.},
doi = {10.1021/pr0503230},
journal = {Journal of Proteome Research},
number = 2,
volume = 5,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}
  • Rhodopseudomonas palustris is among the most metabolically versatile bacteria known. It uses light, inorganic compounds, or organic compounds, for energy. It acquires carbon from many types of green plant-derived compounds or by carbon dioxide fixation, and it fixes nitrogen. Here we describe the genome sequence of R. palustris, which consists of a 5,459,213-base-pair (bp) circular chromosome with 4,836 predicted genes and a plasmid of 8,427 bp. The sequence reveals genes that confer a remarkably large number of options within a given type of metabolism, including three nitrogenases, five benzene ring cleavage pathways and four light harvesting 2 systems. R. palustrismore » encodes 63 signal transduction histidine kinases and 79 response regulator receiver domains. Almost 15% of the genome is devoted to transport. This genome sequence is a starting point to use R. palustris as a model to explore how organisms integrate metabolic modules in response to environmental perturbations.« less
  • Rhodopseudomonas palustris is unique among characterized nonsulfur purple bacteria because of its capacity for anaerobic photoheterotrophic growth using aromatic acids. Like growth with other reduced electron donors, this growth typically requires the presence of bicarbonate/CO{sub 2} or some other added electron acceptor in the growth medium. Proteomic studies indicated that there was specific accumulation of form I ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO) subunit proteins (CbbL and CbbS), as well as the CbbX protein, in cells grown on benzoate without added bicarbonate; such cells used the small amounts of dissolved CO{sub 2} in the medium to support growth. These proteins weremore » not observed in extracts from cells grown in the presence of high levels (10 mM) of added bicarbonate. To confirm the results of the proteomics studies, it was shown that the total RubisCO activity levels were significantly higher (five- to sevenfold higher) in wild-type (CGA010) cells grown on benzoate with a low level (0.5 mM) of added bicarbonate. Immunoblots indicated that the increase in RubisCO activity levels was due to a specific increase in the amount of form I RubisCO (CbbLS) and not in the amount of form II RubisCO (CbbM), which was constitutively expressed. Deletion of the main transcriptional regulator gene, cbbR, resulted in impaired growth on benzoate-containing low-bicarbonate media, and it was established that form I RubisCO synthesis was absolutely and specifically dependent on CbbR. To understand the regulatory role of the CbbRRS two-component system, strains with nonpolar deletions of the cbbRRS genes were grown on benzoate. Distinct from the results obtained with photoautotrophic growth conditions, the results of studies with various CbbRRS mutant strains indicated that this two-component system did not affect the observed enhanced synthesis of form I RubisCO under benzoate growth conditions. These studies indicate that diverse growth conditions differentially affect the ability of the CbbRRS two-component system to influence cbb transcription.« less
  • To assess the applicability of latex cell coatings as an ‘off-the-shelf’ biocatalyst, the effect of osmoprotectants, temperature, humidity and O 2 on preservation of H 2 production in Rhodopseudomonas palustris coatings was evaluated. Immediately following latex coating coalescence (24 h) and for up to 2 weeks of dry storage, rehydrated coatings containing different osmoprotectants displayed similar rates of H2 production. Beyond 2 weeks of storage, sorbitol-treated coatings lost all H 2 production activity, whereas considerable H 2 production was still detected in sucrose- and trehalose-stabilized coatings. The relative humidity level at which the coatings were stored had a significant impactmore » on the recovery and subsequent rates of H 2 production. After 4 weeks storage under air at 60% humidity, coatings produced only trace amounts of H 2 (0–0.1% headspace accumulation), whereas those stored at < 5% humidity retained 27–53% of their H 2 production activity after 8 weeks of storage. In conWhen stored in argon at < 5% humidity and room temperature, R. palustris coatings retained full H 2 production activity for 3 months, implicating oxidative damage as a key factor limiting coating storage. Overall, the results demonstrate that biocatalytic latex coatings are an attractive cell immobilization platform for preservation of bioactivity in the dry state.« less
  • The uptake and anaerobic metabolism of benzoate were studied in short-term experiments with phototrophic cells of Rhodopseudomonas palustris. Cells that were preincubated and assayed anaerobically in the presence of 1 mM dithiothreitol accumulated (7-/sup 14/C)benzoate at a rate of at least 0.5 nmol x min/sup -1/ x mg/sup -1/ of protein. Cells that were preincubated aerobically, or anaerobically in the absence of a reducing agent or an electron donor such as succinate, took up benzoate at reduced rates. Benzoate was removed from the external medium with remarkably high efficiency; initial uptake rates were independent of substrate concentration, and uptake remainedmore » linear down to concentrations of less than 1 ..mu..M. Uptake rates were not sensitive to external pH in the range of 6.5 to 8.1, and very little free benzoate was found associated with the cells. By contrast, benzoyl coenzyme A (CoA) was formed rapidly in cells exposed to labeled benzoate. Its appearance in such cells, together with the more gradual accumulation of other compounds tentatively identified as reduction products, is consistent with the identification of benzoyl CoA as an intermediate in the anaerobic reductive metabolism of benzoate. The very effective uptake of external benzoate can be explained by its conversion to benzoyl CoA immediately after its passage across the cell membrane by simple or facilitated diffusion. Such a chemical conversion would serve to maintain a downhill concentration gradient between the cell cytoplasm and the cell surroundings, even at very low external benzoate concentrations.« less